The present invention is directed to a functional signal anchor that localizes a fusion protein to the apoplast of vascular elements in plants. The signal anchor is useful for engineering secretory proteins to the cell wall and/or apoplast of plant cells. The signal anchor is also useful for producing secretory proteins in transgenic plant cells in a bioreactor.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Xylem and phloem of plant vascular system are major conduits for transportation of water and solutes through the plant (Canny, 1986; Kim and Guerinot, 2007). Xylem and phloem are targets of various kinds of plant pathogens, such as bacteria, fungi and insects. A number of phloem-feeding insects, such as aphids and planthoppers, are highly destructive agricultural pests worldwide (Backus et al 2004, Moran et al, 2001). These pests mainly feed on the stems and suck in the phloem sieve elements, thus, causing direct feeding damage to the crop (e.g. hopper bum) (Moran et al, 2001). These pests also transmit viral diseases that cause additional damages (Noda et al, 1991). Though spraying poisonous chemicals is the usual means used in pest control, which is laborious, expensive and more over not environment-friendly, therefore, use of other safe and economic alternatives of pest control are needed. A number of plant or bacterium derived toxin proteins having insecticidal properties are available and used in transgenic crops (Carlini and Grossi-de-Sa, 2002, Chattopadhyay et al., 2004). The adoption of insect-resistant transgenic crops has been increasing annually ever since the commercial release of the first-generation maize and cotton expressing a single modified Bacillus thuringiensis toxin (Bt) (Christou et al., 2006).
These toxin proteins act differentially against different classes of insects and the toxicity of most of plant derived toxins and Bt to aphids and planthoppers are either unknown or with no effect (Carlini and Grossi-de-Sa, 2002). This is partially due to the fact that the two kinds of insect are phloem-feeding insects, whereas the toxin proteins localize to cytoplasm of plant cell. Similarly, the engineered proteins need to be secreted to plant culture media when transgenic plant cells are used as a natural bioreactor (James and Lee, 2001). In either of the cases, efficient secretion of the engineered proteins to apoplast of plant cells should be considered.
Plants do allow the cost-effective production of recombinant proteins on an agricultural scale, while eliminating risks of product contamination with endotoxins or human pathogens (Fischer and Emans, 2000; Giddings et al., 2000; Ma et al., 2003; Twyman et al., 2003). Plant suspension cells can be employed as host cells for the production of foreign proteins. The main advantages of using transgenic plant cells are due to the fact that the plant culture media is inexpensive and simple. The mammalian proteins produced from plant cells were found to be correctly glycosylated and secreted into the medium (James and Lee, 2001).
Plant disease resistance (R) genes confer race-specific resistance to pathogens that have cognate avirulence (avr) genes (Flor, 1971). The R protein presumably functions as part of a receptor complex that recognizes an elicitor, which is directly or indirectly encoded by the cognate avr gene in the pathogen, and subsequently initiates defense responses (Hammond-Kosack and Jones, 1997; Martin et al., 2003). In recent years, extensive molecular and genetic analyses have been performed in a number of R-Avr systems. The majority of R proteins fall into five classes based primarily upon their combination of a limited number of structural motifs while a few other R proteins have novel structures or confers resistance to plant pathogens in a non-race-specific way (Dangl and Jones, 2001; Martin et al., 2003).
One of the interesting aspects of R protein function is its localization. R proteins have been found in a variety of cellular locations. The available information suggests that R proteins in general colocalize with pathogen effectors, indicating a clear display of spatial interdependency of both components (Martin et al., 2003). The direct physical interactions of R and Avr proteins have been demonstrated in several R-Avr pairs (Jia et al., 2000; Kim et al., 2002; Leister and Katagiri, 2000; Scofield et al., 1996; Tang et al., 1996). Viral effectors are present inside the plant cell, and the predicted structures of all known R proteins against viruses indicate that they are also intracellular (Burch-Smith et al, 2007). The tomato Cf proteins, which recognize extra-cellular Cladosporium fulvum Avr proteins (Lauge and De Wit, 1998), are localized to the plasma membrane (Rivas and Thomas, 2005). Fungal pathogen-directed R proteins can also be intracellular as fungal Avr proteins are delivered to and function inside plant cells (Jia et al., 2000).
All bacteria-directed R proteins are predicted to be intracellular, except XA21. This prediction is based on the fact that most of the bacterial Avr gene products are effector proteins, which are secreted to host cells through the bacterial type III secretion system (TTSS) (He et al., 2004). In fact, many R proteins do not carry recognizable subcellular targeting signatures and their localization needs to be determined experimentally. For instance, Arabidopsis RPM1 and RPS2 are associated with cellular membranes although they do not possess any canonical membrane targeting domains (Axtell and Staskawicz, 2003; Boyes et al., 1998). This subcellular localization is consistent with the membrane localization of their corresponding Avr elicitors, AvrRpm1 and AvrRpt2, respectively (Axtell and Staskawicz, 2003; Nimchuk et al., 2000). Apart from the plasma membrane, Arabidopsis RRS1-R and its cognate Avr protein PopP2 colocalize in the nucleus and the nuclear localization of RRS1-R is dependent on the presence of PopP2 (Deslandes et al., 2003). Recently, both tobacco N and barley MLA10 were found to localize to cytoplasm and nucleus, and nuclear retention of either R protein is indispensable for downstream signaling and defense (Burch-Smith et al., 2007; Shen et al., 2007). In these three cases, translocation of the R proteins during signaling might take place as well upon activation of the R proteins by the cognate Avr proteins (Burch-Smith et al., 2007; Deslandes et al., 2003; Shen et al., 2007). Rice XA21 is a transmembrane receptor kinase that presumably recognizes elicitor localized to apoplast of rice cells with its extracellular LRR portion (Song et al., 1995). The AvrXa21 molecule(s) corresponding to Xa21 has not yet been identified, although it appears that it might be a sulphated protein secreted to the apoplast through a type II secretion system and involved in quorum sensing (Lee et al., 2006).
The apoplast is the extraprotoplastic matrix of plant cells, consisting of all compartments from the external face of the plasmalemma to the cell wall (Dietz, 1997). The apoplast is important for all the plant's communication to its environment and plays an important role in signaling and defense upon pathogen attack (Dietz, 1997; Huckelhoven, 2007). Many extracellular enzymes and proteins located in apoplast or associated with cell wall are involved in signaling for defense or have antimicrobial function (Edreva, 2005; Huckelhoven, 2007). For example, the apoplast contains several low-molecular-weight and protein antioxidants, which control levels of reactive oxygen species (ROS) (Noctor et al., 2002; Pignocchi and Foyer 2003). Apoplastic levels and redox status of ascorbate and glutathione change during compatible and incompatible interactions of barley with B. graminis and several extracellular antioxidative enzyme activities also increase upon B. graminis attack (Noctor et al., 2002; Vanacker et al., 1998, 2000). The increase of peroxidase activity in extracellular space and accumulation of cationic peroxidase in xylem vessels was also detected during interaction of rice with X. oryzae pv oryzae, especially with incompatible interaction (Hilaire et al., 2001; Reimers et al., 1992. Young et al., 1995). Another group of proteins that are secreted to cell wall are pathogenesis-related (PR) proteins. The PR proteins include PR-1, chitinase, glucanases, proteases, thionins, osmotins, defensins, and some of them are only small peptides (Edreva, 2005; Huckelhoven, 2007). These PR proteins induced in resistant or systemic acquired resistance (SAR)-expressing plants, as well as from transgenic resistant plants exhibit high antimicrobial activity (Edreva, 2005). Most of the PR proteins are also induced by many environmental and developmental stimuli (Edreva, 2005).
Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases of rice (Mew 1987). We previously reported isolation of resistance gene Xa27 from rice (Gu et al., 2005). Unlike other cloned R genes, Xa27-mediated resistance specificity to bacterial blight is determined by its promoter rather than by its gene product. Xa27-dependent resistance is associated with the specific induction of the R gene by incompatible pathogens harboring avrXa27. Ectopic expression of Xa27 coding region under rice PR1 promoter resulted in non-specific resistance to both incompatible and compatible strains. The Xa27 protein (XA27) has no sequence similarity with any previously characterized R-gene products.
There is a need to identify elements that are useful for localizing fusion proteins to specific locations within a plant or plant cell.